Although it has been known for over 25 years that development and metamorphosis in insects is under neuroendocrine control, the chemistry, titer, site of synthesis and mode of action of the principal neurohormone involved, the prothoracicotropic hormone (PTTH), has not been elucidated. The paucity of information on PTTH is due in part to the lack of an adequate assay for this putative neuropeptide hormone. Our laboratory has recently developed a sensitive, specific and reproducible in vitro assay for PTTH which should overcome this limitation. In vitro culture techniques for the prothoracic glands and a radioimmunoassay (RIA) for ecdysone have facilitated the establishment of the assay which measures in vitro activation of ecdysone synthesis by the prothoracic glands in response to PTTH. This assay is not only an excellent qualitative method for demonstrating PTTH activity, but when a dose response protocol for PTTH activation of the prothoracic glands is employed, the assay can be utilized for precise quantitation of activity as well. With the in vitro assay a study can now be made of the basic endocrinology and the chemical nature of this neuro-hormone, requisite for future research on the regulation of its synthesis, release and its mode of action. The primary focus of this proposal is the chemical characterization of PTTH. At present there appear to be two molecular forms of the hormone, a low molecular weight peptide and a large one, possibly a glycopeptide. The glycopeptide form of PTTH will be identified via chemical analysis of neutral sugar constituents, radiosynthesis and affinity chromatography. Simultaneously, the peptide form of the hormone will be purified, using the in vitro assay to monitor PTTH activity, and the purified hormone then sequenced, permitting its organic synthesis. With pure hormone a PTTH antibody will then be generated for the development of a PTTH RIA.